Skip to main content
SpringerLink
Log in

The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2

  • Original Article
  • Published:
Protoplasma Aims and scope Submit manuscript

Abstract

Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

CrtRb:

β-Carotene hydroxylase

DiOC6 :

3,3′-Dihexyloxacarbocyanine iodide

GFP:

Green fluorescent protein

HPLC:

High-performance liquid chromatography

PG:

Plastoglobuli

PSY:

PHYTOENE SYNTHASE

RFP:

Red fluorescent protein

References

  • Acevedo F, Rubilar M, Shene C, Navarrete P, Romero F, Rabert C, Jolivet P, Valot B, Chardot T (2012) Seed oil bodies from Gevuina avellana and Madia sativa. J Agric Food Chem 60:6994–7004

    Article  PubMed  CAS  Google Scholar 

  • Al-Babili S, Hartung W, Kleinig H, Beyer P (1999) CPTA modulates levels of carotenogenic proteins and their mRNAs and affects carotenoid and ABA content as well as chromoplast structure in Narcissus pseudonarcissus flowers. Plant Biol 1:607–612

    Article  CAS  Google Scholar 

  • Bhaskar PB, Venkateshwaran M, Wu L, Ané J-M, Jiang J (2009) Agrobacterium mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato. PLoS One 4:e5812

    Article  PubMed  Google Scholar 

  • Bionda T, Tillmann B, Simm S, Beilstein K, Ruprecht M, Schleiff E (2010) Chloroplast import signals: the length requirement for translocation in vitro and in vivo. J Mol Biol 402:510–523

    Article  PubMed  CAS  Google Scholar 

  • Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C (2002) Membrane protein transport between the endoplasmic reticulum and the golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching. Plant Cell 14:1293–1309

    Article  PubMed  CAS  Google Scholar 

  • Breithaupt DE, Bamedi A (2002) Carotenoids and carotenoid esters in potatoes (Solanum tuberosum L.): new insights into an ancient vegetable. J Agric Food Chem 50:7175–7181

    Article  PubMed  CAS  Google Scholar 

  • Britton G (1995) Structure and properties of carotenoids in relation to function. FASEB J 9:1551–1558

    PubMed  CAS  Google Scholar 

  • Brown CR, Kim TS, Ganga Z, Haynes K, De Jong D, Jahn M, Paran I, De Jong W (2006) Segregation of total carotenoid in high level potato germplasm and its relationship to beta-carotene hydroxylase polymorphism. Am J Pot Res 83:365–372

    Article  CAS  Google Scholar 

  • Cazzonelli CI (2011) Carotenoids in nature: insights from plants and beyond. Func Plant Biol 38:833–847

    Article  CAS  Google Scholar 

  • Cazzonelli CI, Pogson BJ (2010) Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci 15:266–274

    Article  PubMed  CAS  Google Scholar 

  • Cho HB, Winans SC (2005) VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals. Proc Natl Acad Sci USA 102:14843–14848

    Article  PubMed  CAS  Google Scholar 

  • Cookson PJ, Kiano JW, Shipton CA, Fraser PD, Romer S, Schuch W, Bramley PM, Pyke KA (2003) Increases in cell elongation, plastid compartment size and phytoene synthase activity underlie the phenotype of the high pigment-1 mutant of tomato. Planta 217:896–903

    Article  PubMed  CAS  Google Scholar 

  • D'Ambrosio C, Giorio G, Marino I, Merendino A, Petrozza A, Salfi L, Stigliani AL, Cellini F (2004) Virtually complete conversion of lycopene into beta-carotene in fruits of tomato plants transformed with the tomato lycopene beta-cyclase (tlcy-b) cDNA. Plant Sci 166:207–214

    Article  Google Scholar 

  • Diretto G, Al-Babili S, Tavazza R, Papacchioli V, Beyer P, Giuliano G (2007) Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway. PLoS One 2:e350

    Article  PubMed  Google Scholar 

  • Ducreux LJM, Morris WL, Hedley PE, Shepherd T, Davies HV, Millam S, Taylor MA (2005) Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein. J Exp Bot 56:81–89

    PubMed  CAS  Google Scholar 

  • Ducreux LJM, Morris WL, Prosser IM, Morris JA, Beale MH, Wright F, Shepherd T, Bryan GJ, Hedley PE, Taylor MA (2008) Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes. J Exp Bot 59:4219–4231

    Article  PubMed  CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984

    Article  PubMed  CAS  Google Scholar 

  • Fraser PD, Bramley PM (2004) The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265

    Article  PubMed  CAS  Google Scholar 

  • Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP, and related tools. Nat Protoc 2:953–971

    Article  PubMed  CAS  Google Scholar 

  • Fraser PD, Enfissi EMA, Bramley PM (2009) Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches. Arch Biochem Biophys 483:196–204

    Article  PubMed  CAS  Google Scholar 

  • Fraser PD, Truesdale MR, Bird CR, Schuch W, Bramley PM (1994) Carotenoid biosynthesis during tomato fruit development. Plant Physiol 105:405–413

    PubMed  CAS  Google Scholar 

  • Fray RG, Wallace A, Fraser PD, Valero D, Hedden P, Bramley PM, Grierson D (1995) Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway. Plant J 8:693–701

    Article  CAS  Google Scholar 

  • Galpaz N, Wang Q, Menda N, Zamir D, Hirschberg J (2008) Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J 53:717–730

    Article  PubMed  CAS  Google Scholar 

  • Giuliano G, Tavazza R, Diretto G, Beyer P, Taylor M (2008) Metabolic engineering of carotenoid biosynthesis in higher plants. Trends in Biotech 26:139–145

    Article  CAS  Google Scholar 

  • Hirschberg J (2001) Carotenoid biosynthesis in flowering plants. Curr Opp Plant Biol 4:210–218

    Article  CAS  Google Scholar 

  • Howitt CA, Pogson BJ (2006) Carotenoid accumulation and function in seeds and non-green tissues. Plant Cell Environ 29:435–445

    Article  PubMed  CAS  Google Scholar 

  • Iwanzik W, Tevini M, Stute R, Hilbert R (1983) Carotenoid content and composition of different potato varieties and their importance for the color of the flesh of the tuber. Pot Res 26:149–162

    Article  CAS  Google Scholar 

  • Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium mediated plant transformation. Trends Plant Sci 7:193–195

    Article  PubMed  CAS  Google Scholar 

  • Kessler F, Schnell D, Blobel G (1999) Identification of proteins associated with plastoglobules isolated from pea (Pisum sativum L.) chloroplasts. Planta 208:107–113

    Article  PubMed  CAS  Google Scholar 

  • Koning AJ, Lum PY, Williams JM, Wright R (1993) DIOC6 staining reveals organelle structure and dynamics reveals organelle structure and dynamics in living yeast cells. Cell Mot Cytoskel 25:111–128

    Article  CAS  Google Scholar 

  • Kovacs K, Zhang L, Linforth RST, Whittaker B, Hayes CJ, Fray RG (2007) Redirection of carotenoid metabolism for the efficient production of taxadiene taxa-4(5),11(12)-diene in transgenic tomato fruit. Trans Res 16:121–126

    Article  CAS  Google Scholar 

  • Li FQ, Vallabhaneni R, Yu J, Rocheford T, Wurtzel ET (2008) The maize phytoene synthase gene family: overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and thermal stress tolerance. Plant Physiol 147:1334–1346

    Article  PubMed  CAS  Google Scholar 

  • Li H-M, Chiu C-C (2010) Protein transport into chloroplasts. In S Merchant, WR Briggs, D Ort, eds, Ann Rev Plant Biol 61: 157–180

  • Li L, Paolillo DJ, Parthasarathy MV, Dimuzio EM, Garvin DF (2001) A novel gene mutation that confers abnormal patterns of beta-carotene accumulation in cauliflower (Brassica oleracea var. botrytis). Plant J 26:59–67

    Article  PubMed  CAS  Google Scholar 

  • Lopez AB, Eck JV, Conlin BJ, Paolillo DJ, O'Neill J, Li L (2008) Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers. J Exp Bot 59:213–223

    Article  PubMed  CAS  Google Scholar 

  • Morris WL, Ducreux L, Griffiths DW, Stewart D, Davies HV, Taylor MA (2004) Carotenogenesis during tuber development and storage in potato. J Exp Bot 55:975–982

    Article  PubMed  CAS  Google Scholar 

  • Morris WL, Ducreux LJM, Fraser PD, Millam S, Taylor MA (2006) Engineering ketocarotenoid biosynthesis in potato tubers. Metab Eng 8:253–263

    Article  PubMed  CAS  Google Scholar 

  • Naested H, Holm A, Jenkins T, Nielsen HB, Harris CA, Beale MH, Andersen M, Mant A, Scheller H, Camara B, Mattsson O, Mundy J (2004) Arabidopsis VARIEGATED 3 encodes a chloroplast targeted, zinc-finger protein required for chloroplast and palisade cell development. J Cell Sci 117:4807–4818

    Article  PubMed  CAS  Google Scholar 

  • Orzaez D, Mirabel S, Wieland WH, Granell A (2006) Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant Physiol 140:3–11

    Article  PubMed  CAS  Google Scholar 

  • Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Adams JL, Silverstone AL, Drake R (2005) Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotech 23:482–487

    Article  CAS  Google Scholar 

  • Pringle JR, Preston RA, Adams AEM, Stearns T, Drubin DG, Haarer BK, Jones EW (1989) Fluorescence microscopy methods for yeast. Meths Cell Biol 31:357–435

    Article  CAS  Google Scholar 

  • Quinlan RF, Shumskaya M, Bradbury LM, Beltran J, Ma C, Kennelly EJ, Wurtzel ET (2012) Synergistic interactions between carotene ring hydroxylases drive lutein formation in plant carotenoid biosynthesis. Plant Physiol 160:204–214

    Article  PubMed  CAS  Google Scholar 

  • Rabbani S, Beyer P, Von Lintig J, Hugueney P, Kleinig H (1998) Induced beta-carotene synthesis driven by triacylglycerol deposition in the unicellular alga Dunaliella bardawil. Plant Physiol 116:1239–1248

    Article  PubMed  CAS  Google Scholar 

  • Rey P, Gillet B, Romer S, Eymery F, Massimino J, Peltier G, Kuntz M (2000) Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress. Plant J 21:483–494

    Article  PubMed  CAS  Google Scholar 

  • Rochasosa M, Sonnewald U, Frommer W, Stratmann M, Schell J, Willmitzer L (1989) Both developmental and metabolic signals activate the promoter of a class I patatin gene. EMBO J 8:23–29

    CAS  Google Scholar 

  • Ruiz-Sola MA, Rodriguez-Concepcion M (2012) Carotenoid biosynthesis in Arabidopsis: a colorful pathway. Arabidopsis Book/Am Soc Plant Biol 10:e0158

    Google Scholar 

  • Santos CAF, Senalik D, Simon PW (2005) Path analysis suggests phytoene accumulation is the key step limiting the carotenoid pathway in white carrot roots. Gen Mol Biol 28:287–293

    Article  CAS  Google Scholar 

  • Shumskaya M, Bradbury LM, Monaco RR, Wurtzel ET (2012) Plastid localization of the key carotenoid enzyme phytoene synthase is altered by isozyme, allelic variation, and activity. Plant Cell 24:3725–3741

    Article  PubMed  CAS  Google Scholar 

  • Shumskaya M, Wurtzel ET (2013) The carotenoid biosynthetic pathway: thinking in all dimensions. Plant Science 208:58–63

    Article  PubMed  CAS  Google Scholar 

  • Siddique MA, Grossmann J, Gruissem W, Baginsky S (2006) Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts. Plant Cell Physiol 47:1663–1673

    Article  PubMed  CAS  Google Scholar 

  • Steinmüller D, Tevini M (1985) Composition and function of plastoglobuli. Planta 163:201–207

    Article  PubMed  Google Scholar 

  • Thomson WW, Platt K (1973) Plastid ultrastructure in the barrel cactus, Echinocactus acanthodes. New Phytol 72:791–797

    Article  Google Scholar 

  • Tian L, DellaPenna D (2004) Progress in understanding the origin and functions of carotenoid hydroxylases in plants. Arch Biochem Biophys 430:22–29

    Article  PubMed  CAS  Google Scholar 

  • Weisman LS, Emr SD, Wickner WT (1990) Mutants of Saccharomyces cerevisiae that block intervacuole vesicular traffic and vacuole division and segregation. Proc Natl Acad Sci USA 87:1076–1080

    Article  PubMed  CAS  Google Scholar 

  • Ye XD, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303–305

    Article  PubMed  CAS  Google Scholar 

  • Ytterberg AJ, Peltier JB, van Wijk KJ (2006) Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes. Plant Physiol 140:984–997

    Article  PubMed  CAS  Google Scholar 

  • Zhang R, Wise RR, Struck KR, Sharkey TD (2010) Moderate heat stress of Arabidopsis thaliana leaves causes chloroplast swelling and plastoglobule formation. Photosynth Res 105:123–134

    Article  PubMed  CAS  Google Scholar 

  • Zhou X, McQuinn R, Fei Z, Wolters AA, Van Eck J, Brown C, Giovannoni JJ, Li L (2011) Regulatory control of high levels of carotenoid accumulation in potato tubers. Plant Cell Environ 34:1020–1030

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by the Scottish Government Rural and Environment Science and Analytical Services Division and EU-FP7 METAPRO 244348.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK

    Stefania Pasare, Kathryn Wright, Raymond Campbell, Wayne Morris, Laurence Ducreux, Sean Chapman, Alison Roberts & Mark Taylor

  2. School of Biological Sciences, Royal Holloway, University of London, Egham Hill, Egham, Surrey, TW20 OEX, UK

    Stefania Pasare, Peter Bramley & Paul Fraser

Authors
  1. Stefania Pasare
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kathryn Wright
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Raymond Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wayne Morris
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Laurence Ducreux
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sean Chapman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peter Bramley
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Paul Fraser
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alison Roberts
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mark Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark Taylor.

Additional information

Handling Editor: Jaideep Mathur

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pasare, S., Wright, K., Campbell, R. et al. The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2. Protoplasma 250, 1381–1392 (2013). https://doi.org/10.1007/s00709-013-0521-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-013-0521-z

Keywords

Search

Navigation